For purposes of the present disclosure, the term “pulse interval” means the time from the beginning of one pulse to the beginning of the next pulse.
The following publications are discussed hereinbelow:
U.S. Pat. No. 6,010,613;
U.S. Pat. No. 6,603,998;
U.S. Pat. No. 6,713,291;
“Enhancement of Cellular Immune Response to a Prostate Cancer DNA Vaccine by Intradermal Electroporation”, by Roos et al, Molecular Therapy, Vol. 13, No. 2, February 2006, pages 320-327 (referred to herein as Roos et al);
“The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy”, by Pucihar et al, Bioelectrochemistry 57 (2002) pages 167-172 (referred to herein as Pucihar et al).
Vernhes M C, Cabanes P A, Tessie J. Chinease hamster ovary cells sensitivity to localized electrical stress. Bioelectrochemistry and Bioenergetics. 1999, 48:17-25;
Daskalov I, Mudrov N, Peycheva E. Exploring new instrumentation. Parameters for electrochemotherapy. Attacking tumors with bursts of biphasic pulses instead of single pulses. 1999, IEEE Eng. Med. Biol 62-66; Chang D C, Cell poration and cell fusion using an oscillating electric field. 1989 Biophys J. 56:641-652; and
Tekle E, Astumian R D, Chock P B. Electroporation by using bipolar oscillating electric field: An improved method for DNA transfection of NIH 3T3 cells. 1991 Proc. Natl. Acad. Sci. 88:4230-4234.
U.S. Pat. No. 6,010,613, incorporated herein by reference, discloses using electroporation with wide interval electrical waveforms, such as provided by PA-4000 System (referred to herein as PulseAgile) of Cyto Pulse, Inc., 810 Cromwell Park Drive, Suite T, Glen Burnie, Md. 21061. More specifically, U.S. Pat. No. 6,010,613 discloses applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, to a material, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses.
For purposes of the discussions and disclosures herein, the above-mentioned applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, to a material, with the characteristics (1), (2), and (3) set forth is referred to herein as “PulseAgile”.
The specification disclosed in U.S. Pat. No. 6,010,613 and the documentation connected with the PulseAgile system provide that the pulse interval is equal to or greater than 0.1 seconds, which is 100 milliseconds. Hereinafter, the PulseAgile generated electrical waveforms which have pulse intervals which are equal to or greater than 100 milliseconds are referred to as “wide interval PulseAgile electrical waveforms” or “slow PulseAgile electrical waveforms”.
In U.S. Pat. No. 6,010,613, there is no specific evidence presented that administered vaccines have either successful genetic expression of the vaccine or provide improved T-cell response involving improved secretion of good protein resulting from successful genetic expression of the vaccine.
Both U.S. Pat. No. 6,603,998 and U.S. Pat. No. 6,713,291, both incorporated herein by reference, disclose the delivery of polynucleotide vaccines to biological cells using the wide interval PulseAgile electrical waveforms or slow PulseAgile electrical waveforms.
Roos et al disclose the use of the wide interval PulseAgile electrical waveforms or slow PulseAgile electrical waveforms to deliver a polynucleotide vaccine into mammalian skin cells. It is also disclosed by Roos et al that successful genetic expression of the polynucleotide vaccine is demonstrated by detection of a genetic marker which expresses luciferase protein. In addition, Roos et al disclose that with the use of the wide interval PulseAgile electrical waveforms or the slow PulseAgile electrical waveforms to deliver a polynucleotide vaccine into mammalian skin cells, there is improved T-cell response involving improved secretion of good protein resulting from successful genetic expression of the polynucleotide vaccine. In Roos et al, T-cell response is represented by PSA-specific IFN(gamma)-producing CD8+ T cells.
Aside from the beneficial results disclosed in the Roos et al publication, there are two undesirable results observed by using the slow PulseAgile electrical waveforms. The first undesirable result is that each slow PulseAgile electrical waveform administration protocol took approximately 3.5 seconds. Since administration employing the use of needles penetrating into mammalian skin causes discomfort or pain, for such 3.5 second administration protocol, the mammal would have to endure the discomfort or pain for approximately 3.5 seconds.
The second undesirable result disclosed in Roos et al is that each slow PulseAgile electrical waveform causes a perceptible muscle contraction. The muscle contraction itself can also cause discomfort or pain. Normally, for an administration of a polynucleotide vaccine, plural pulsed waveforms would be applied to a mammal. Therefore, plural muscle contractions, with plural additional muscle discomfort or pain, would take place with such slow PulseAgile electrical waveforms.
Pucihar et al disclose that, before their publication date in 2002, electrical pulses have been used in combination with chemotherapeutic agents to treat cancerous cells. The earlier electrical pulses have had a frequency of 1 Hz, whereby each pulse produced a related tetanic contraction (muscle contraction). It is noted that 1 Hz translates to 1000 milliseconds per cycle. The discussed electrical pulse protocols are all pulse sequences that have pulses of uniform pulse amplitude, uniform pulse width, and uniform pulse interval. The chemotherapeutic agents include small nonpermeant hydrophilic molecules. The disclosures of the research conducted by Pucihar et al relate to in vitro (not in vivo) experiments with cancerous cell being treated with Lucifer Yellow, which is a small nonpermeant hydrophilic molecule. The disclosures of the research conducted by Pucihar et al explore various pulse repetition frequencies in order to exceed the frequency of tetanic contraction (so that successive muscle contractions fuse into smooth motion). There is a statement in Pucihar et al that with a frequency of excitation of 40 Hz or faster, successive muscle contractions fuse into smooth motion. The 40 Hz pulse frequency employs pulses of uniform pulse amplitude, uniform pulse width, and uniform pulse interval. It is noted that 40 Hz translates to 25 milliseconds per cycle.
Vernhes et al disclose that viability and permeability of CHO cells electroporated in vitro were high over an electroporation pulse frequency range of 0.5 to 100 HZ.
Daskalov et al disclose that eight bipolar pulses delivered to tumor cells in vivo produced a similar response to electrochemotherapy when delivered at 1 HZ and 1 kHZ.
Chang discloses that high frequency sinusoidal waveforms delivered as short pulses efficiently electroporated COS-M-6 cells in vitro.
Tekle et al disclose that unipolar or bipolar rectangular wave pulses delivered at frequencies ranging from 60 kHZ to 1 MHZ efficiently transfected NIH 3T3 cells in vitro.
There is no disclosure in any of Pucihar, Vernhes et al, Daskalov et al, Chang, or Tekle et al which states any relationship to polynucleotide vaccination, to successful genetic expression of a polynucleotide vaccine, or to improved T-cell response involving improved secretion of a desired protein resulting from successful genetic expression of the polynucleotide vaccine.
In view of the above, it would be desirable to provide a method and apparatus for the delivery of polynucleotide vaccine into mammalian skin cells which takes less than 3.5 seconds to administer the polynucleotide vaccine.
In addition, it would be desirable to provide a method and apparatus for the delivery of polynucleotide vaccines into mammalian skin cells which applies plural PulseAgile electrical waveforms to the mammalian skin and only causes one muscle contraction for the plural applied electrical waveforms.
Administration of a polynucleotide vaccine, to be successful, must give evidence of successful genetic expression of the administered polynucleotide vaccine. Moreover, to be successful, the genetic expression of the administered polynucleotide must give evidence of providing a desired protein which results from the successful genetic expression of the polynucleotide vaccine.
Thus, while the foregoing body of prior art indicates it to be well known to use electroporation apparatuses, the prior art described above does not teach or suggest a method and apparatus for the delivery of polynucleotide vaccines into mammalian skin cells which has the following combination of desirable features: (1) provides a method and apparatus for the delivery of polynucleotide vaccine into mammalian skin cells which takes less than 3.5 seconds to administer the polynucleotide vaccine; (2) applies plural PulseAgile electrical waveforms to the mammalian skin and only causes one muscle contraction for the plural applied electrical waveforms; (3) gives evidence of successful genetic expression of the administered polynucleotide vaccine; and (4) gives evidence of providing a desired protein which results from the successful genetic expression of the polynucleotide vaccine. The foregoing desired characteristics are provided by the unique method and apparatus for the delivery of polynucleotide vaccines into mammalian skin cells of the present invention as will be made apparent from the following description thereof. Other advantages of the present invention over the prior art also will be rendered evident.
The foregoing desired characteristics are provided by the unique method and apparatus for the delivery of polynucleotide vaccines into mammalian skin cells of the present invention as will be made apparent from the following description thereof. Other advantages of the present invention over the prior art also will be rendered evident.